Our research is aimed at the design and synthesis of small molecule modulators of Histone Acetyltransferase (HAT) enzymes. The paucity of potent and selective small molecule HAT modulators has limited our understanding of the biological functions of this protein family. We are focused specifically on members of the GNAT and MYST families, as there is a wealth of high-resolution structural information available for several important members. Additionally, genetic studies suggest that selective small molecule modulators of these HATs may have potential therapeutic applications in the treatment of a variety of cancers and metabolic disorders. We have synthesized a structurally diverse library of molecules and developed a robust biochemical assay for HAT activity. The structures of the GNAT and MYST active sites demonstrated that each contains a cysteine residue, although the position of the cysteine is distinct in the two families. We have therefore initially focused our synthetic efforts on electrophilic small molecules, which have the potential to interact with the active site cysteine. We have synthesized libraries of reversible and irreversible electrophilic small molecules, and are currently screening these in vitro for modulation of HAT activity. Initial hits will be further developed to increase potency and specificity. We are developing a series of clickable HAT probes based on the electrophilic core structure and using these in human cells and lysates to better understand their modes and mechanisms of action. A recent study revealed a novel regulatory region of a human HAT protein outside of the canonical catalytic domain. We are currently extending these studies to understand this regulatory mechanism and further optimize our small molecule modulators. We are also developing derprobes for specific human HAT activities. These probes are constructed from modular protein domains that undergo a conformational switch in response to lysine acetylation. This change in conformation is measured indirectly using Frster Resonance Energy Transfer (FRET). These probes are suitable for in vitro and cell-based studies of HAT activity. The domain structure of the probes will be varied to provide simultaneous read-out of multiple HAT activities within the same cell. Fluorescence imaging techniques are used to visualize and quantify relative HAT activity under a variety of cellular conditions and treatments. These are being developed for the eventual application to cell-based screening for novel molecular modulators of HAT and HDAC pathways.